13C glucose breath test for the diagnosis of diabetic indications and monitoring glycemic control

ABSTRACT

Use of  13 C glucose in an analytical assay to monitor glucose metabolism by measurement of labeled exhaled CO 2  is provided. A breath test and kit for performing the breath test are described for the diagnosis of diabetic indications and monitoring of glycemic control. The breath test utilizes the measurement of expired  13 C-labeled CO 2  following the ingestion of a  13 C-enriched glucose source.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/439,290,filed May 16, 2003, now U.S. Pat. No. 6,878,550, which is a continuationof application Ser. No. 10/228,281, filed Aug. 27, 2002, now U.S. Pat.No. 6,599,750, which is a continuation of application Ser. No.09/910,701, filed Jul. 20, 2001, now U.S. Pat. No. 6,461,870, which is acontinuation-in-part of U.S. application Ser. No. 09/674,806, now U.S.Pat. No. 6,468,802, filed Feb. 8, 2001, as a U.S. National PhaseApplication of International Application PCT/IB99/00933, filed May 9,1999 which claims benefit of U.S. Provisional Application No.60/084,482, filed May 6, 1998. The disclosure of each of the aboveapplications is incorporated herein by reference in their entirety.

FIELD OF INVENTION

Use of ¹³C glucose in an analytical assay to monitor glucose metabolismby measurement of labeled exhaled CO₂ is provided. A breath test and kitfor performing the breath test are described for the diagnosis ofdiabetic indications and monitoring of glycernic control. The breathtest utilizes the measurement of expired ¹³C-labeled CO₂ following theingestion of a ¹³C-enriched glucose source.

REFERENCES

The following references are referred to by their numbers in parenthesisin this specification.

-   -   1. Martin B C, Warram J H, Krolewski A S, et al. Role of glucose        and insulin resistance in development of type 2 diabetes        mellitus: results of a 25-year follow-up study. Lancet 1992;        340: 925–9.    -   2. Lillioja S, Mott D M, Spraul M, et al. Insulin resistance and        insulin secretory dysfunction as precursors of        non-insulin-dependent diabetes mellitus; prospective studies of        Pima Indians. N Engl J Med 1993; 329: 1988–92.    -   3. Beck Nielsen H, Groop L C. Metabolic and genetic        characterization of pre-diabetic states. Sequence of events        leading to non-insulin-dependent diabetes mellitus. J Clin        Invest 1994; 94: 1714–21.    -   4. Matthaei S, Stumvoll M, Kellerer M, et al. Pathophysiology        and pharmacological treatment of insulin resistance. Endocr Rev        2000; 21: 585–618.    -   5. The Expert Committee on the Diagnosis and Classification of        Diabetes Mellitus. Report of the expert committee on the        diagnosis and classification of diabetes mellitus. Diabetes Care        2001; 24(suppl 1).    -   6. Harris M I. Undiagnosed NIDDM: clinical and public health        issues. Diabetes Care 1993, 16: 642–52.    -   7. World Health Organization. Prevention of diabetes mellitus:        report of a WHO study group. Geneva: WHO, 1994; technical report        series No. 844.    -   8. Meltzer S, Leiter L, Daneman D, et al. 1998 clinical practice        guidelines for the management of diabetes in Canada. Can Med        Assoc J 1998; 159 (suppl 8): S1–29.    -   9. Matthews D R, Hosker J P, Rudenski A S, et al. Homeostasis        model assessment: insulin resistance and beta-cell function from        fasting plasma glucose and insulin concentrations in man.        Diabetologia 1985; 28: 412–19.    -   10. Haffner S M, Gonzales C, Miettinene H, et al. A prospective        analysis of the HOMA model: the Mexico City Diabetes Study.        Diabetes Care 1996: 19: 1138–41.    -   11. Bonora E, Targher G, Alberiche M, et al. Homeostasis model        assessment closely mirrors the glucose clamp technique in the        assessment of insulin sensitivity. Diabetes Care 2000; 23:        57–63.    -   12. World Health Organization. Definition, diagnosis and        classification of diabetes mellitus and its complications:        Report of a WHO Consultation. Part 1. Diagnosis and        classification of diabetes mellitus. Geneva: WHO, 1999.    -   13. Ganda O P, Day J L, Soeldner J S, et al. Reproducibility and        comparative analysis of repeated intravenous and oral glucose        tolerance tests. Diabetes 1978; 27:715–25.    -   14. Riccardi G, Vaccaro O, Rivellese A, et al. Reproducibility        of the new diagnostic criteria for impaired glucose tolerance.        Am J Epidemiol 1985; 121: 422–9.    -   15. Ko G T C, Chan J C N, Woo J, et al. Use of the 1997 American        Diabetes Association Diagnostic criteria for diabetes in a Hong        Kong Chinese population. Diabetes Care 1998; 21: 2094–7.    -   16. Ko G T C, Chan J C N, Woo J, et al. The reproducibility and        usefulness of the oral glucose tolerance test in screening for        diabetes and other cardiovascular risk factors. Ann Clin Biochem        1998; 35: 62–7.    -   17. Gabir M M, Hanson R L, Diabelea D, et al. The 1997 American        Diabetes Association and 1999 World Health Organization criteria        for hyperglycemia in the diagnosis and prediction of diabetes        Diabetes Care 2000; 23: 1108–12.    -   18. Radziuk J. Insulin sensitivity and its measurement:        structural commonalities among the methods. J Clin Endocrinol        Metab 2000; 85: 4426–33.    -   19. CDC Diabetes Cost-Effectiveness Study Group. The        cost-effectiveness of screening for type 2 diabetes. JAMA 1998;        280: 1757–63.    -   20. Hosker J P, Matthews D R, Rudneski A S, et al. Continuous        infusion of glucose with model assessment: measurement of        insulin resistance and β-cell function in man. Diabetologia        1985; 28: 401–11.    -   21. Emoto M, Kawagishi T, Nishizawa Y, et al. Homeostasis model        assessment as a clinical index of insulin resistance in type 2        diabetic patients treated with sulfonylureas. Diabetes Care        1999; 22:818–22.

The entire disclosure of each of the above-referenced publications,patents and patent applications is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Glucose tolerance is defined as the ability to properly utilize glucose.Diabetes is not a single disease, but an array of diseases that exhibitthe common symptom of glucose intolerance, an impairment in glucoseutilization.

The prevalence of diabetes in the general population is approximately6–7%. Only about half of diabetics are actually diagnosed. Studies haveshown that rates for persons with glucose intolerance are equal by sexand greater for blacks than for whites.

In general, the following types of diabetes have been recognized: type Idiabetes mellitus, type II diabetes mellitus, secondary diabetesmellitus, impaired glucose tolerance and gestational glucose mellitus.The general characteristics of the symptoms of diabetes include thefollowing:

-   -   Polyuria (high urine blood volume)    -   Hyperglycemia (high blood glucose levels)    -   Glucosuria (loss of glucose in urine)    -   Polydipsia (excessive thirst)    -   Polyphagia (excessive hunger)    -   Sudden weight loss

It has been observed that complications resulting from diabetes mellitusare the third leading cause of death in most developed countries.Diabetes is a risk factor for a variety of conditions including coronaryheart disease, cerebrovascular stroke, neuropathy (nerve damage),nephropathy (kidney damage), retinopathy (eye damage), hyperlipidemia(excessive blood lipids), angiopathy (damage to blood vessels) andinfection.

A number of different methods exist for determining a condition ofintolerance for glucose. These include postprandial blood glucose, oralglucose tolerance test (OGTT), O'Sullivan glucose tolerance test(gestational test), hemoglobin Alc (Hb A^(l), Hb A_(lc)), islet cellantibodies, glutamic acid decarboxylase (GAD) antibodies and insulinantibodies. Diabetes, however, is most readily detected when thecarbohydrate metabolic capacity is tested. This is done by stressing thesystem with a defined glucose load as in the oral glucose tolerance test(OGTT).

The OGTT has been criticized, however, because many of the variablesaffecting test results are difficult to control. For instance: patientsmust be on a standardized carbohydrate diet at least three days beforethe test; the test requires an 8 to 16 hour fast; the test should onlybe performed on ambulatory patients; stress should be avoided; exerciseshould be avoided; various hormone imbalances can affect validity suchas with: thyroxine, growth hormone, cortisol and catecholamines; variousdrugs and medications can affect validity such as: oral contraceptives,salicylates, nicotinic acid, diuretics and hypoglycemics; and evaluationshould normally be corrected for age. The greatest disadvantage of theOGTT is that it is poorly reproducible and this limits its diagnosticusefulness.

Type 2 diabetes is a common condition, associated with significantmorbidity and mortality. It is generally acknowledged that overt type 2diabetes is preceded by a period of glucose intolerance which itself ispreceded by a significant period of insulin resistance (1–5). It is nowfurther recognized that typical diabetic complications can begin todevelop during this “pre-diabetic” phase (3,6). The identification ofpersons at risk of developing overt type 2 diabetes has therefore takenon even greater importance. It has been suggested that if such personscould be easily identified, a lifestyle modification strategy could beimplemented which might prevent their progression to type 2 diabeteswith its attendant morbidities.

Because of the public health importance of type 2 diabetes, regularscreening for this condition is now advocated (5,7,8). However, suchscreening programs, whether by fasting plasma glucose or by the 75-gOGTT, only identify diabetic or glucose-intolerant patients. Thehomeostasis model assessment (HOMA) index has been advocated as a methodof detecting persons with insulin resistance and therefore presumably atrisk of progressing to overt type 2 diabetes (9–11). However, the HOMAindex requires a serum insulin measurement and, some argue, the use of acomputer program. Thus, this index is not as simple or accessible as afasting blood glucose level. Similarly, the gold standard euglycemic,hyperinsulinemic clamp is clearly not appropriate for mass screeningcampaigns.

The current methods of diagnosing diabetes involve either invasivetesting (i.e., repeated blood collections), or use blood-borne markers(i.e., glycosylated proteins, or antibodies) which offer an indirectassessment of glucose regulation. Accordingly, it is an object of thepresent invention to avoid the need for invasive testing or the use ofblood-borne markers in determinations of glucose regulation.

SUMMARY OF THE INVENTION

The above and other objects of the invention are attained by a ¹³Cbreath test and a kit for determining glucose regulation in a patient inneed thereof.

Based on our experience in the use of ¹³C breath tests, we propose asimple, sensitive test of insulin resistance. In normal individuals, inthe presence of insulin, glucose is taken up by cells where it undergoesglycolysis and then enters the citric acid cycle or is shunted to fatsynthesis. In either case, CO₂ is produced as a metabolic by-product.This CO₂ then re-enters the circulation and is eliminated in the lungs.We found that if glucose was labeled with ¹³C, the resultant CO₂ couldbe detected in the expired air. In type 2 diabetes and other states ofinsulin resistance, glucose uptake is impaired and the generation of¹³CO2 is likewise blunted. Accordingly, we have developed a ¹³C-glucosebreath test for the diagnosis of type 2 diabetes and insulin resistance.In particular, the test provides a means to detect insulin resistancewhen blood glucose levels are still in the normal range and beforeβ-cell destruction leading to diabetes has occurred. Early detection ofinsulin resistance will allow intervention in time to prevent thedevelopment of type 2 diabetes. In addition, the test allows the successof intervention therapies, including diet and exercise. to be monitored.

An analytical assay is described that is based on the use ofnon-radioactive ¹³C. Labeled expired ¹³CO₂ is measured in the presentassay. Isotope ratio mass spectroscopy (IRMS) is used as a detectionmethod for ¹³C, a non-radioactive isotope that occurs naturally in foodand animal tissues. Non-dispersive infrared spectroscopy (NDIRS)analysis and analysis methods known in the art may be employed. The testprotocol is as follows: after an overnight fast, the oral dose of ¹³Cuniformly labeled glucose (containing about 25 mg of ¹³C glucose incombination with about 15 g of unlabeled glucose in 100 ml of tap water)is administered. Breath samples will be collected before the dose andthen 1½ hours after ¹³C glucose ingestion. Levels of ¹³CO₂ in expiredair will be measured by an IRMS method.

Advantages of this test are the following:

-   -   it is practical, sensitive and specific;    -   the validity of the test is not influenced by stress, exercise,        hormone imbalances, or some drugs and medications;    -   it is a non-invasive method;    -   it is simple to perform and can be readily used in physicians        offices or medical laboratories;    -   it is safe since ¹³C is a naturally occurring isotope found in        all carbon-containing substances;    -   it involves no radioactivity, and may be used in children and        women.

The ¹³C glucose test is safe, reliable, and specific in diagnosis ofdiabetes and measurement of the severity of insulin resistance inpatients. The invention is also preferred to diagnose gestationaldiabetes and to monitor glycemic control in diabetes patients. Apreferred embodiment of the invention is a kit containing the necessarymaterial for performing the described method. This kit may contain, butis not limited to, a source of ¹³C enriched glucose (preferablyuniformly labeled D-glucose); a source of unenriched glucose; and abreath collection device. The kit may also contain a set of patientinstructions for its use. In another embodiment, the kit mayadditionally contain a blood collection device, such as a lancet orhypodermic needle and vacutainer for the additional determination ofblood glucose levels.

Accordingly, in one aspect the invention provides diagnostic kits forthe determination of glycemic control in a subject comprising: apredetermined quantity of ¹³C-enriched glucose; and a breath collectioncontainer. A plurality of breath containers and/or instructions for usemay be included. The kits may be used for the diagnosis of diabetes,insulin resistance, gestational diabetes, and the like or to determinethe adequacy of antihyperglycemic therapy.

In a further aspect, the invention provides a use of ¹³C-enrichedglucose for the determination of glycemic control in a subject.

In another aspect, the invention provides ¹³C-enriched glucose for usein the manufacture of diagnostic kits for the determination of glycemiccontrol in a subject. The kits may be used for the diagnosis ofdiabetes, insulin resistance, gestational diabetes, and the like or todetermine the adequacy of antihyperglycemic therapy.

In yet a further aspect, the invention provides diagnostic kits for thedetermination of glycemic control in normal, diabetic and insulinresistant subjects by comparing blood glucose levels with breath levelsof ¹³C-enriched CO₂

In a still further aspect, the invention provides method of diagnosing acondition in a subject, said condition selected from the groupconsisting of diabetes, insulin resistance impaired glucose tolerance,impaired fasting glucose and gestational diabetes, said methodcomprising collecting a first breath sample from said subject in a firstbreath collection container; administering ¹³C-enriched glucose to saidsubject; collecting a second breath sample from said subject in a secondbreath container at a time point after administration of said¹³C-enriched glucose; measuring the ¹³CO₂ in each of said first andsecond breath samples; and comparing the amount of ¹³CO₂ in said secondbreath sample with the amount of ¹³CO₂in said first breath sample toobtain a delta value, wherein the presence of less ¹³CO₂ in said secondbreath sample compared to normal control values indicates the presenceof said condition. Using an ROC curve, a delta cutoff is chosen whereinthe sensitivity and specificity are such as to maximize diagnosticaccuracy. In particular, when the condition is insulin resistance, arange of deltas from 8 to 10 is preferred. A delta of 9 is mostpreferred.

In yet an additional aspect, the invention provides method of predictinga subject's risk of developing diabetes, said method comprisingcollecting a first breath sample from said subject in a first breathcollection container; administering ¹³C-enriched glucose to saidsubject; collecting a second breath sample from said subject in a secondbreath container at a time point after administration of said¹³C-enriched glucose; measuring the ¹³CO₂ in each of said first andsecond breath samples; and comparing the amount of ¹³CO₂ in said secondbreath sample with the amount of ¹³CO₂ in said first breath sample,wherein the presence of less ¹³CO₂ in said second breath sample comparedto normal control values indicates risk of developing diabetes. Thecomparison may be made by choosing a cutoff of ROC values wherein thesensitivity and specificity are such as to maximize diagnostic accuracy.In particular, a range of ROC's from 8 to 10 is preferred. An ROC of 9is most preferred.

The ¹³C-glucose breath test is superior to currently used laboratorycriteria in the diagnosis of type 2 diabetes. Its predictive value forclinical status, as well as its correlation with the HOMA index, make ita simple but useful test for detecting early evidence of insulinresistance and hence, risk for type 2 diabetes.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1: Illustrates the IRMS analysis of ¹³C glucose breath samples fromnormal individuals, a gestational diabetic, and patients with impairedglucose tolerance.

FIG. 2: Shows a representative example of breath test and blood glucoselevels of a normal individual.

FIG. 3: Illustrates breath test and blood glucose levels of a diabeticpatient.

FIG. 4: Depicts breath test and blood glucose levels of an insulinresistant patient.

FIG. 5: Shows a comparison of IRMS results of an insulin resistant and adiabetic patient, and a normal individual.

FIG. 6: Illustrates the distribution of ¹³CO₂ (a) and HOMA (b) resultsbetween diabetic and non-diabetic patients. Bars indicate the upperlimit of the normal range.

FIG. 7: Plots the relationship between HOMA index and ¹³C-glucose breathtest result.

FIG. 8: Illustrates the ROC curve for the ¹³C-glucose breath test.Individual points are in δ per mil.

DETAILED DESCRIPTION OF THE INVENTION

The introduction of a ¹³C breath test offers a novel, non-invasive,direct means to monitor glucose metabolism by measurement of exhaled CO₂using highly enriched, uniformly labeled ¹³C-glucose. Glucose metabolismwill generate labeled CO₂, which is then exhaled and collected in tubes.Enrichment of labeled CO₂, over a determined time course, can be used asa quantitative index of glucose metabolism. Comparison is made againstage-specific reference intervals.

The present invention has a number of advantages, including lower doseof glucose needed (overcomes inconsistencies due to malabsorptivedisorders or previous gastric or intestinal surgery), reduction intesting time (from the current 2 hours required for the OGTT) and fewerinterpretational ambiguities (greater sensitivity and specificity).

The ¹³C glucose breath test is based on the metabolism of glucose.Following a baseline breath sample, a ¹³C glucose solution containingabout 25 mg of ¹³C glucose in combination with about 15 g of unlabeledglucose in 100 ml of tap water is administered. Breath samples will beobtained before the dose and then 12 hours after ¹³C glucose ingestion.Measurement of the expired air will be detected by an isotope ratio massspectroscopy assay method. Elevated or excessive breath of ¹³CO₂concentrations will be seen in individuals who have normal glucosemetabolism.

The ¹³C-glucose breath test provides a more sensitive and diagnosticallyaccurate indicator of the presence of type 2 diabetes than do currentlyused common methodologies. However, a problem arises in that thedefinition of diabetes is made on the basis of fasting plasma glucose orglucose-tolerance test values. Thus, these tests are the defacto “goldstandards” and theoretically should be the most accurate. In thewell-characterized group of diabetic patients studied in thisinvestigation, the pitfalls of a single fasting blood glucose value or aglucose tolerance test are evident. Indeed, numerous reports of the pooroverall diagnostic accuracy of the glucose tolerance test or fastingplasma glucose as a diagnostic tool for diabetes exist (13–17).Moreover, the requirement for confirmation of an abnormal fasting plasmaglucose reduces sensitivity of this test albeit at a gain inspecificity. It could be argued, however, that for screening purposes,sensitivity is perhaps preferable to specificity. However, because ofthe theoretical advantage of diagnosing subjects at risk of diabetesprior to the actual onset of the disease, various indices of insulinresistance or glucose intolerance have been devised (for a review see18). The hypothesis associated with these latter measurements is thatinsulin resistance and abnormalities in glucose homeostasis occur wellbefore the onset of overt type 2 diabetes. If patients demonstratingsuch abnormalities can be detected through screening programs, it hasbeen suggested that the development of overt diabetes may be preventedor delayed (4,5,19). The importance of such an approach is furtherunderscored by the finding that at the time of type 2 diabetes onset, asignificant number of patients already have diabetic complications(3,6).

In order to address the need for a relatively simple index of insulinresistance, the HOMA index was developed. This index has been shown tocorrelate with results from the gold-standard hyperinsulinemic,euglycemic clamp (9,11,20,21). Although the HOMA index was significantlyhigher in the diabetics in this study, it was diagnostically inferior inall aspects to the ¹³C-glucose breath test. Indeed, when both the HOMAindex and the ¹³C-glucose breath test results were entered into alogistic regression which included fasting blood sugar, age, sex and BMIas variables, only the ¹³C-glucose breath test gave a statisticallysignificant partial correlation coefficient. Similarly, when each of thetwo variables of interest was individually included in a similarlogistic regression, which also included the 2 hour OGTT value as afurther variable, the ¹³C-glucose breath test retained a statisticallysignificant predictive value whereas the HOMA index did not. Indeed, inall possible iterations of the logistic regression, the ¹³C-glucosebreath test was always the strongest predictor of diabetic status.Although it may be argued that a HOMA is an easier test, requiring onlya single blood sample, there are disadvantages to this test as well.First of all, a serum insulin measurement must be carried out in areasonably advanced medical laboratory by trained technicians. This addstime and cost to the screen. The ¹³C-glucose breath test, however, canbe analyzed using a point of care instrument that requires very littletraining to use. Thus, screening can be carried out in the field withresults available almost as soon as the last breath sample is complete.The HOMA index requires blood samples with the attendant infectiousprecautions. The ¹³C-glucose breath test is carried out on breath andtherefore only general infectious precautions are necessary. Similarly,phlebotomy requires trained medical personnel whereas the ¹³C-glucosebreath test does not necessarily require any supervision—a packageinsert can provide all the necessary instructions. Thus, the ¹³C-glucosebreath test can also be made available to remote locations via post.Finally, although the HOMA provides added diagnostic accuracy to thediagnosis of diabetes when compared to a fasting blood sugar, as can beseen from Table 1, the traditional OGTT is superior to both. Compared tothe OGTT, however, the ¹³C-glucose breath test has even greater accuracyand has the advantage of requiring a lower glucose load and a shortertime requirement along with all the other advantages listed above. Onefinal consideration is the possibility of false negative results withthe breath test in subjects with delayed gastric emptying. Given therelatively low volume and lower osmolarity of the breath test comparedwith the OGTT, problems with gastric emptying are likely to be less thanthose associated with the OGTT. Indeed, based on 1, 1.5 and 2 hourbreath test values in this study, no subjects showed evidence of delayedgastric emptying. As this test is most likely to find use early in thecourse of insulin resistance/type 2 diabetes, it is unlikely thatdiabetic gastroparesis will be a significant confounder. Thus, the¹³C-glucose breath test offers a simple, sensitive and accurate methodfor the diagnosis of type 2 diabetes.

In terms of insulin resistance, studies are underway to validate the¹³C-glucose breath test against the hyperinsulinemic, euglycemic clamp.However, even with the current results, there is evidence that the¹³C-glucose breath test is an indicator of insulin resistance. First,the ¹³C-glucose breath test results do correlate with the HOMA.Secondly, there is a strong correlation between the breath test and bodymass index whereas the correlation between the HOMA index is lessstrong. Third, the superior diagnostic parameters of the breath test andthe fact that a type 1 diabetic had a breath result of <1.2 show acorrelation between insulin resistance and the ¹³C-glucose breath testresult. Finally, the underlying principal of the ¹³C-glucose breath testis based on resistance to glucose uptake by target tissues. Thus, the¹³C-glucose breath test also offers a simple, sensitive, specific testfor the diagnosis of insulin resistance.

One final advantage of the ¹³C-glucose breath test is its applicationfor following insulin resistance. This test has the potential to allowthe effectiveness of various interventions in type 2 diabetes to bemonitored. Whether these interventions be lifestyle or pharmacological,the ¹³C-glucose breath test offers a sensitive, dynamic method to assesseffectiveness of type 2 diabetes treatments.

Thus, the ¹³C-glucose breath test may be used not only to diagnosediabetes, but also to determine insulin sensitivity and insulinresistance. The test may reliably be used to diagnose other difficult todetect pre-diabetic conditions. Thus, it is a useful tool to determinewhether a patient is at risk of developing diabetes.

It is important that any diagnostic test procedure have diagnostic.accuracy, i.e., that it accurately predicts positive and negativevalues. The receiver operated characteristics (ROC) value describes thebalance between the sensitivity (i.e., the number of hits detected) andthe specificity (i.e., the accuracy) of a test. These two variables mayalso be considered positive predictive value and negative predictivevalue, and are correlated with diagnostic accuracy. The ROC curve showsthe relationship of the probability of a positive test, given nodisease, to the probability of a positive test, given disease. An ROCcutoff value is chosen to maximize diagnostic accuracy of the test inquestion.

The following examples serve to illustrate the present invention. Theseexamples are not intended to limit the scope of the invention in anymanner.

EXAMPLE 1 Sample Assay for Diagnosis of a Patient

Experimental Procedure

Medical History

Medical history is taken and includes, but is not limited to: theabsence of active pulmonary disease, no history of heart, liver, orrenal failure, and no use of insulin or oral medications for thetreatment of diabetes.

Physical Examination and Laboratory Tests

No physical examination or laboratory tests, including blood sampling,is required.

Dietary Control

It is determined that all participants have fasted overnight prior tocommencement of the test.

Patient Control

Participants are not permitted to eat, drink, or smoke during the test.All patients are required to remain sedentary for the duration of thetest. Small amounts of water are allowed.

Assay Procedure

Patients fast for at least 8 hours before this test.

A sample set of patient instructions is given below:

Step 1: Collect First Breath Sample

-   -   Remove the screw cap from the collection tube.    -   Take a normal breath and then exhale fully 4 to 8 seconds        through a straw into the bottom of the collection tube.    -   Immediately replace the screw cap on the collection tube and        tighten until snug (do not overtighten).    -   Affix the completed green label to the collection tube.

Step 2: Drink the Solution

-   -   Prepare the solution by adding tap water to the fill line on the        plastic container. Mix until completely dissolved and then drink        the entire solution.    -   Wait 1½ hours.

Step 3: Collect the Second Breath Sample

-   -   One and one half hours after drinking the solution, collect the        second breath sample into the collection tube following the same        directions as for the first breath sample in step 1.    -   Affix the completed yellow label to the tube.

Step 4: Return the Samples for Analysis

-   -   Insert the 2 collection tubes along with the signed and        completed registration card in the mailing box.    -   Return the mailing box as instructed to the site of dispensing.

EXAMPLE 2 Breath Test Administration

Patients are given an exetainer tube with the screw cap removed. Usingthe straw, they are asked to breathe into the tube, exhaling normally,for 4 to 8 seconds. Next, each patient is instructed to drink a solutioncontaining about 25 mg of uniformly labeled ¹³C glucose in combinationwith about 15 g of unlabeled glucose in 100 ml of tap water. After 12hours, the patients are given a new tube to breathe in as describedabove. The breath collection is then complete.

Storage and Shipping

Breath test tubes are typically labeled with the patient's name andidentification number and shipped to an analytical laboratory foranalysis. No refrigeration or special storage techniques are necessary.

EXAMPLE 3 Analytical Methodology

Breath specimens are analyzed by isotope ratio mass spectroscopy. NDIRSis also a preferred method to analyze breath test samples. Other methodsknown in the art may also be used.

Statistical Analysis

The sensitivity, specificity, positive and negative predictive values ofthe breath test are compared to that of the oral glucose tolerance test.Receiver operated characteristic curve (ROC) analysis is performed toconfirm the discrimination between type 2 diabetes or gestationaldiabetes and individuals with normal glucose metabolism.

EXAMPLE 4 Basis of the Method of IRMS

Isotope ratio mass spectroscopy (IRMS) is a highly precise method ofanalysis which is able to measure small samples (low nanogram amounts).For example, ¹³C/¹²C ratios are determined on a mono-carbon molecule;CO₂ gas. The CO₂ gas can be directed to the spectrometer by means of acontinuous flow IRMS (also called CF-IRMS).

The statistical combination of the isotopes of carbon (¹²C and ¹³C) andoxygen (¹⁶O, ¹⁷O, ¹⁸O) to generate the CO₂ molecules gives rise to theformation of various isotopomers whose molecular weights are 44, 45, and46, respectively. Thus, for measuring carbon isotope ratios, 3 ion beamsare generated and recorded in the IRMS, corresponding to the masses ofthe various isotopomers of CO₂.

In order to obtain a high precision and a high accuracy, reference gasesof absolutely known isotopic composition are used and a dual inletsystem allows an alternative admission of both sample and referencegases into the ionization source via a gas-switching valve. Themeasurement of the various ion beams allows for the calculation of the¹³C enrichment of the sample. The value of this calculation is givenδ¹³C(‰) notation. The ¹³C abundance is expressed as δ¹³C(‰) according tothe following:δ¹³ C(‰)=([¹³ C/ ¹² C)sample/(¹³ C/ ¹² C)PDB]−1)×1000

This δ¹³C(‰) value measures the variations in parts per thousand of thecarbon isotope ratio from the standard. For carbon, PDB was selected asthe international reference. PDB is Pee Dee Belemnitella (a fossil fromthe Pee Dee geological formation in South Carolina). The ¹³C/¹²C ratiofrom the calcium carbonate of this fossil is 0.011237. Compared to PDB,most of the natural compounds display a negative delta value. In theabove equation, ¹³C/¹²C refers to the isotopomers.

Using the breath test of this invention, IRMS is an example method todiagnose type 2 and gestational diabetes, and for monitoring glycemiccontrol of diabetes patients.

EXAMPLE 5 ¹³C Glucose Breath Test Results of Normal, GestationalDiabetes and Impaired Glucose Tolerance Patient

Example 4 describes a method to analyze breath samples of thisinvention. FIG. 1 shows the mean (±SD) Delta per mil over Baseline (DOB)of the normal population. Also shown are the DOB's of a gestationaldiabetic and impaired glucose tolerance patients. Breath samplescollected 0, 1, 1.5 and 2 hours according to the protocol were analyzedby IRMS. IRMS analysis of the collected breath samples can be performedon various instruments including, but not limited to, the AP2003 andAP2002 (Analytical Precision Ltd.), ABCA (POZ Europa) and the Breath MAT(Finnigan MAT). The DOB values of the gestational diabetes and theimpaired glucose tolerance patients are well below the DOB of the normalpopulation (FIG. 1). The impaired glucose tolerance diagnosis wasinitially determined by OGTT, the gestational diabetes screen was usedto confirm gestational diabetes.

Impaired glucose tolerance (IGT) refers to a condition in which bloodsugar levels are higher than normal, but are not high enough to beclassified as diabetes. IGT is a major risk factor for type 2 diabetes.IGT is present in about 11 percent of adults, or approximately 20million Americans. About 40–45 percent of persons age 65 years of age orolder have either type 2 diabetes or IGT. A person is currentlydiagnosed with IGT when the 2-hour glucose results from a glucosetolerance test are greater than 7.8 mmol/L, but less than 11.0 mmol/L. Awoman is diagnosed with gestational diabetes when she is pregnant andhas any two of the following: a fasting plasma glucose of more than 5.3mmol/L, a 1-hour glucose level of more than 10.6 mmol/L, a 2-hourglucose level of more than 8.9 mmol/L. However, as this method ofdiagnosis is invasive, the breath tests of the current invention is thepreferred diagnosis method. The ¹³C glucose breath test is sensitive,accurate and non-invasive.

EXAMPLE 6 ¹³C Glucose Breath Test Results of a Normal, Insulin Resistantand Diabetes Patient

In this example, both breath test and blood glucose levels were done ona normal, diabetic and insulin resistant patient. FIG. 2 shows the DOBof 0, 1, 1.5 and 2 hours breath samples of a normal subject analyzed byIRMS. The blood glucose level of this normal individual is alsodisplayed.

FIG. 3 illustrates the breath test and blood glucose levels of adiabetic patient. The DOB of the breath samples are significantly lowerthan the DOB of the normal individual (FIG. 2), the blood glucose levelsare typical of a diabetic patient.

In FIG. 4, the breath test and blood glucose levels of aninsulin-resistant patient are depicted. The DOB of these breath samplesare significantly lower than the normal DOB (FIG. 2), the blood glucoselevels are typical of an insulin-resistant patient.

These results demonstrate one preferred utility of the breath test ofthe current invention to diagnose diabetes and insulin resistance. Inanother aspect of the invention, the areas between the breath test andblood glucose test curves can be used to diagnose patients with insulinresistance or diabetes and confirm glucose tolerance in normalindividuals by the comparison of the areas to the different groups ofnormal, diabetic and insulin resistant patients.

FIG. 5 illustrates the ¹³C glucose breath test results of a normalindividual, insulin resistant and diabetes patient. The DOB's of theinsulin resistant and diabetes patients is significantly lower than thatof the normal DOB results.

EXAMPLE 7 NDIRS Instrumentation

Breath test samples of the invention can also be analyzed using NDIRSinstrumentation. The course of the ¹³C₂/¹²CO₂ ratio in breath allows fordiagnosis of diabetes. NDIRS can be further used to diagnose type 2 andgestational diabetes patients and for monitoring therapy of diabetespatients (glycemic control of these patients).

The metabolism of ¹³C labeled substrate leads to a different isotoperatio. NDIRS analysis of the invention can be performed on variousinstruments, including, but not limited to, the MicroLyzer (QuinTron),UbiT-IR200 and UbiT-100 (Otsuka Pharmaceutical Co., Ltd.), the URAS 10(Hartmann and Braun) and the Isomax 2000 (Isotechnika).

EXAMPLE 8 Hyperinsulinemic Euglycemic Clamp Method for the Measurementof Insulin Resistance

Insulin resistance is defined as the decrease of the biological actionof insulin, and it mainly presents as an hyperinsulinemia. Thehyperinsulinemic euglycemic clamp is currently the reference method forquantifying insulin resistance. The clamp technique consists of infusinginsulin at a constant rate and, to prevent any decrease in the plasmaglucose level, by infusing dextrose. The rate of dextrose infused tomaintain euglycemia is an estimate of the amount of glucose, which istaken up by the tissues under the effect of a defined plasma insulinconcentration. Using several rates of insulin infusion allows theestablishment of the relationship between the whole body glucosedisposal and plasma insulin levels, and to discriminate between thestates of decreased insulin sensitivity and/or altered maximal capacityto dispose of glucose. However, the hyperinsulinemic euglycemic clampmethod is very invasive, time consuming, costly and variable. The breathtest of this invention is a preferred method to measure insulinresistance as it is reliable, sensitive, specific, cost-effective andnon-invasive.

EXAMPLE 9 Monitoring Long-Term Control of Diabetes

Measuring glycated hemoglobin is a current test used for monitoringlong-term control of diabetes. Glycated hemoglobins are increased as areflection of hyperglycemia during the life span of erythrocytes.However, different analytical methods may measure different glycatedhemoglobins and caution must be exercised in the interpretation ofresults. HPLC or column chromatography methods used to analyze glycatedhemoglobin are also highly sensitive to variations in temperature andpH. This test is also invasive, requiring several blood samples. Thebreath test of the present invention is preferred as it is non-invasive,sensitive, accurate and cost-effective.

EXAMPLE 10 Usefullness of ¹³C Glucose Breath Test in Diagnosis ofDiabetes

Diabetes mellitus is a group of diseases characterized by high levels ofblood glucose resulting from defects in insulin secretion, insulinaction, or both. Diabetes can be associated with serious complicationsand premature death if left undiagnosed and untreated. It has beenestimated by the World Health Organization that the number of peoplesuffering from diabetes worldwide will more than double from about 135million now to 300 million by the year 2025. Of those estimated to havediabetes, it is believed that approximately one third of those areundiagnosed. It is also known that the prevalence of diabetes increaseswith age. It is estimated that 0.16% of people under the age of 20 havediabetes but this number dramatically increases to 18.4% for people overthe age of 65.

There are four types of diabetes; type 1 (insulin dependent) represents5 to 10% of all diagnosed cases, type 2 (non-insulin-dependent diabetes)represents 90 to 95% of all diagnosed cases, gestational diabetesdevelops in 2 to 5% of all pregnancies but disappears when a pregnancyis over, and other specific types of diabetes resulting from specificgenetic syndromes, surgery, drugs, malnutrition, infections and otherillnesses may account for 1 to 2% of all diagnosed cases. A number ofdifferent methods exist for determining diabetes. These includepostprandial blood glucose, oral glucose tolerance test (OGTT),O'Sullivan glucose tolerance test (gestational test), hemoglobin Alc,islet cell antibodies, glutamic acid decarboxylase (GAD) antibodies, andinsulin antibodies. However, diabetes is most readily detected when thecarbohydrate metabolic capacity is tested. This is done by stressing thesystem with a defined glucose load as in the OGTT.

Although the OGTT is a standard test for diabetes, it has beencriticized because many of the variables affecting the test results aredifficult to control for; the standardized carbohydrate diet, eight tosixteen hour fast, stress, exercise, hormone imbalances, and variousdrugs can cause test variables. These variables lead to poorreproducibility and limit the diagnostic usefulness of this test. Inaddition, the OGTT involves the collection of numerous blood specimensmaking it an invasive procedure.

The development of a ¹³C-glucose breath test for the detection ofdiabetes offers a non-invasive method that is not affected by the abovementioned variables. ¹³C is a non-radioactive isotope that occursnaturally in food and animal tissues. In the past the disadvantage of¹³C had been the shortage of the gas isotope mass spectrometers used foranalysis. With the ready availability of the necessary instrumentationand the ¹³C-labeled compounds required, the use of ¹³C-labeled compoundsin breath tests is more feasible.

Clinical Study

Objective: The primary aim of this pilot study is to evaluate thesensitivity, specificity and reliability of a ¹³C-D-glucose breath testin the diagnosis of type 2 and gestational diabetes as compared to thealready validated glucose tolerance test that will be considered thestandard.

Design: A multi-center, blinded, non-randomized design is utilized. Onlythe referring physicians have knowledge of the participants' status.Participants undergo a glucose tolerance test. Within two weeksfollowing, participants undergo a ¹³C-D-glucose breath test. Thefindings from both tests are examined for concordance.

Study Participants: This investigation is carried out by recruiting 50individuals each for type 2 and gestational diabetes. For type 2diabetes, the participants are suspected to be diabetic. For gestationaldiabetes, the participants are women in their 24^(th) to 28^(th) week ofpregnancy who have presented for the standard gestational diabetesmellitus screening test. Any diagnosis of diabetes is based on theresults of the glucose tolerance test.

Testing Strategy: Eligible participants, after giving informed consent,undergo the glucose tolerance test and the ¹³C-D-glucose breath testseparated by a minimum of 24 hours and a maximum of two weeks. Theglucose tolerance test is performed according to the guidelines of theCanadian Diabetes Association (CMAJ, JAMC Oct. 20, 1998;159(8suppl):S1-S29). Briefly, for the gestational diabetes screen, theglucose tolerance test consists of the consumption of a 50 g glucosetolerance drink and the collection of a venous blood sample one hourlater for glucose determination. For the time between the drinkconsumption and the blood sampling, the participant remains sedentaryand refrains from smoking or eating. Small sips of water may be taken ifnecessary.

For type 2 diabetes, an overnight fast (10–16 hours) precedes theglucose tolerance test. A fasting glucose blood sample is drawn prior tothe consumption of a 75 g glucose tolerance drink. Two hours after theingestion of the drink, a venous blood sample is collected for glucosedetermination. For the time between the drink consumption and the bloodsampling, the participant remains sedentary and refrains from smoking oreating. Small sips of water may be taken if necessary.

The ¹³C-D-glucose breath test is preceded by an overnight fast (minimumeight hours). After fasting, the participants are required to provide abaseline breath sample. The participants then ingest the ¹³C-D-glucosedrink preparation and will provide breath samples at 1, 1.5, and 2hours. During the test the participants remain sedentary and are notpermitted to smoke or eat. Only small sips of water are permitted duringthe test.

Overall Study Design: A total of 50 participants are investigated eachfor type 2 and gestational diabetes.

Visit One: During the recruitment process, each individual is asked toreview a Participant Information Sheet and to talk with the laboratorypersonnel to ensure that all eligibility requirements are met. Theindividual is given an opportunity to ask questions and if they meet allthe eligibility criteria, they are asked to read and sign and InformedConsent Form.

All participants who have met the eligibility criteria and signed aconsent form are tested by both the glucose tolerance test (Visit Two)and the ¹³C-D-glucose breath test (Visit Three) separated by a minimumof 24 hours and a maximum of two weeks.

Visit Two: The glucose tolerance test follows the guidelines set out bythe Canadian Diabetes Association (CMAJ, JAMC Oct. 20, 1998;159(8suppl):S1-S29). Briefly for the gestational diabetes screen, theparticipants are asked to consume a commercially available glucosetolerance drink consisting of 50 g of dextrose in 296 ml. One hourfollowing consumption, a venous blood sample is collected into ared-topped vacutainer tube. For type 2 diabetes, participants firstcomplete and overnight fast (10–16 hours) and then provide a fastingblood glucose sample. Participants then ingest a commercially availableglucose tolerance drink consisting of 75 g of dextrose in 296 mlfollowed by the collection of a venous blood sample 2 hourspost-consumption.

Visit Three: For the ¹³C-D-glucose breath test, participants firstcomplete an overnight fast (minimum of 8 hours). Participants provide abaseline breath sample which is followed by consumption of a¹³C-D-glucose-enriched solution containing 25 mg of ¹³C-D-glucose incombination with 15 g of unlabeled USP dextrose in 100 ml of water.

Participants then provide breath samples at 1, 1.5, and 2 hours.

Note: Visit One and Visit Two may be combined if it is more convenientand all the testing criteria are met.

NUMBER OF PARTICIPANTS AND TARGET POPULATION: A total of 100 adultparticipants (18 years of age or older) who are suspected of having type2 diabetes (n=50) or are being screened for gestational diabetes (n=50)are recruited from those individuals presenting for the oral glucosetolerance test.

INTERIM ANALYSIS: After 25 participants are enrolled for a particulartype of diabetes, all parties are unblinded to the participants status.At this point in the study, the results are evaluated. If the¹³C-D-glucose breath test results do not correlate with the standard,the oral glucose tolerance test, such that greater than 5% of theparticipants are reported as false negatives or false positives, thestudy is temporarily halted. If the study is halted, the protocol isamended to reflect an adjustment in the ¹³C-D-glucose breath test kitcomponents such that it contains 50 mg of ¹³C-D-glucose and 15 g ofunlabeled USP dextrose.

EXAMPLE 11 Advantages of the ¹³C Glucose Test for the Diagnosis ofDiabetes

The disadvantages of the OGTT include uncontrollable factors which causevariability or spurious results and the invasiveness of the test. Othertests known in the are not specific, are invasive, are variable and arelabor intensive. The ¹³C glucose breath test of the present invention issensitive, reliable and specific. The ¹³C glucose breath test showsminimal intra-individual variation, excellent analytical precision andbreath specimens are stable for at least six weeks at room temperature.The ¹³C glucose breath test is preferred over tests known in the art, itis non-invasive, easy to perform, has very good sensitivity andspecificity and is cost effective. A preferred use of the breath test ofthis invention is for the diagnosis of type 2 and gestational diabetes.This invention is also preferred to determine the level of insulinresistance and for monitoring the appropriateness of the therapy ofdiabetes patients.

EXAMPLE 12 Efficacy of the C-Glucose Breath Test in the Detection ofType 2 Diabetes and Insulin Resistance

Fifty-four diabetic subjects, aged 18–75, were recruited from attendeesof the University of Alberta Hospital diabetes education program. Allsuch subjects who took part in this study did so after attending theprogram wherein their diabetic status was verified by an endocrinologistaccording to WHO diagnostic criteria (12). Patients with secondary formsof diabetes or who were on medications which might otherwise interferewith insulin sensitivity were excluded. Similarly, patients in whom a12-hour medication-free run-in period or fast was thought to bemedically contraindicated were excluded from this study. Fifty normalsubjects were recruited from the population-at-large as well as fromspouses of participating diabetic patients. We report results for onlytype 2 diabetics in this study although we have carried out the protocolin a few carefully selected type 1 diabetics for verification. Thisproject was approved by the Research Ethics Board of the University ofAlberta Faculty of Medicine and all subjects gave their informed consentprior to participating in the study.

In this study, each subject underwent breath testing as well as astandard 75 g oral glucose tolerance test (OGTT) in random order.Following a 12-hour overnight fast, study subjects attended theUniversity of Alberta Hospital Metabolic Center at 8:00 a.m. Noanti-diabetic medications, including insulin, were taken within 12 hoursof the study and, in particular, glyburide was not taken within 24 hoursof the study. Subjects were allowed free access to water during thefast, however. At time zero of the standard glucose tolerance test, aserum insulin level was also obtained in a proportion of study subjects.This modification to the protocol was added after a planned interimanalysis indicated enhanced sensitivity of the breath test over OGTTparameters. Plasma glucose was measured by glucose oxidase methodologyand serum insulin was measured by automated immunoassay (Elecsys 2010®,Roche Diagnostic, Basel, Switzerland). For the breath test, subjectsprovided a baseline breath sample and then drank 250 ml of the breathtest solution. Serial breath samples were obtained at 1, 1.5 and 2 hoursafter consumption of the breath test solution. Capillary blood glucose.readings were obtained every 30 minutes during the 2 hours of the breathtest.

The ¹³C breath test consisted of 25 mg of ¹³C-glucose mixed with 15 g ofdextrose and orange flavoring. The ¹³C-glucose (Martek BiosciencesCorporation, Maryland, USA) is universally labeled meaning the ¹³Coccupies all six carbon positions in the molecule. Previous optimizationstudies had demonstrated that 25 mg of ¹³C-glucose was sufficient fordiagnostic purposes and that 15 g of glucose provided an adequatecaloric challenge. In order to carry out the test, a baseline breathsample was obtained followed by breath samples at 1, 1.5 and 2 hoursfollowing the test drink. The expired ¹³CO₂ following test drinkingestion was compared to the baseline value and results expressed as anabsolute increase in ¹³C in δ per mil. Although optimization studiessuggested 1.5 hours as the best time for breath sampling, we included 1and 2 hour time points to verify these findings. Similarly, although aprevious receiver operated characteristics (ROC) suggested 8.5 as thecutoff between normal and abnormal, and a range of useful δs is from 8to 10, we also repeated ROC analysis using the data in this study. Onthis basis a δ per mil of 9.0 was ultimately used as a cutoff betweendiabetic and non-diabetic for this experiment.

¹³CO₂ was measured in breath samples using the AP2003, an isotope ratiomass spectrometer (Analytical Precision Limited, Cheshire, England). Toobtain breath sample, subjects were asked to blow the value of a normalexhalation through a short straw into (10 ml) gas sampling tubes (LabcoExetainer® system—¹³C and gas testing vials, Labco Limited,Buckinghamshire, England). The tubes were then immediately stoppereduntil analyzed. These tubes are known to be impermeable to gases for upto 90 days following sealing. Gas sampling from the tubes occurs via aneedle in the AP2003 machine permeating a rubber membrane present in thecap of the tube. The same apparatus and overall method is commonly usedin other ¹³C breath tests such as the ¹³C urea breath test forHelicobacter pylon.

A 75 gram glucose tolerance test was carried out according to standardprotocol. As mentioned, a baseline fasting serum insulin level wasobtained in a sub-sample of the study population. Glucose was collectedin lithium-heparin tubes and immediately assayed in order to ensure nochanges in apparent glucose concentration. The HOMA index was calculatedas previously described using the formula HOMA=(fasting glucose xfasting insulin)/22.5 (9). A value of 2.5 or above was taken to beindicative of insulin resistance.

All values are expressed as the mean±SD unless otherwise indicated.Sensitivity, specificity, positive predictive value, negative predictivevalue and diagnostic accuracy were calculated according to standardmethodology. Differences in variables between groups were compared usingtwo-sided, unpaired t-tests, ANOVA or ANCOVA as appropriate. Post hoctesting for ANOVA was carried out using the Tukey test. Correlationsbetween variables were carried out using linear regression. Logisticregression was used to determine factors which most accurately predicteddiabetic status. A p value of less than 0.05 was consideredstatistically significant. Statistical analysis was carried out usingStatview version 5.0.1 (SAS Institute, Cary, N.C., USA).

A total of 53 diabetic, 50 normal and 5 subjects with impaired glucosetolerance were included in the primary analysis. Of this total, asubgroup of 45 individuals (21 diabetic, 24 normal) underwentsimultaneous measurement of fasting serum insulin along with fastingplasma glucose in order to calculate a HOMA. Mean age of diabeticpatients was 54±11 and the mean age of normals was 44±14 (p<0.0001 fordifference). Body mass index (BMI) of diabetics was 31.6±5.5 and that ofnormals 28.8±5.2 (p=0.011). Although there were differences betweendiabetics and normals in terms of age and BMI, using age and/or BMI ascovariates did not significantly affect the results.

A scatter diagram for diabetics and normals for both the ¹³C breath testand the HOMA are shown in FIGS. 5 a and 5 b. From this figure, it can beseen that there is significantly less overlap between normals anddiabetics for the ¹³C breath test results than for the HOMA indexresults. Table 1 compares diagnostic variables for the fasting plasmaglucose, OGTT (WHO criteria), the HOMA index and the ¹³C breath test. Asnot all subjects had fasting serum insulin values measured, diagnosticparameters for the ¹³C breath test in the subgroup that did have theirHOMA indexes calculated are presented in a separate column. For thepurposes of this analysis, subject were classified as either diabetic ornon-diabetic based on the criterion of a fasting plasma glucose of 7.0or greater or a 2-hour OCTT value of 11.1 or greater. The classificationof impaired fasting glucose was not used as this diagnosis is notreadily verifiable from clinical data. From this table, it can be seenthat the sensitivity, negative predictive value and overall diagnosticaccuracy of the ¹³C-glucose breath test is superior to that of a fastingplasma glucose, OGTT or HOMA in the diagnosis of type 2 diabetes.Similarly, the positive predictive value and the specificity of the¹³C-glucose is superior to that of the HOMA in making the diagnosis oftype 2 diabetes. These latter two parameters are not applicable to thefasting plasma glucose or OGTT, however, as an abnormality in eithercriterion would change the status of a subject from “normal” to“diabetic”. Thus, based on a single measurement, there can be no falsepositives by fasting plasma glucose or OGTT. Indeed, one supposedlynormal subject did have a fasting plasma glucose of 7.0 but for purposesof this analysis, was still considered “normal”. It is noteworthy thatthis same individual was categorized as “diabetic” on the basis of the¹³C breath test. If agreement between two fasting blood glucoses is usedas a basis for diagnosis, then the estimated sensitivity of a FPG was37%, specificity 82%, PPV 74%, NPV 48% and diagnostic accuracy 55%.However, these values are estimates, using the fasting capillary glucoseas a “second FPG”, taking into account an expected 15% lower glucoseresult for the capillary glucose compared to the FPG. Thus, therequirement for confirmation of any single diagnostic value by a repeatvalue would, as expected, decrease sensitivity but increase specificity.

FIG. 6 demonstrates the relationship between the 1.5-hour ¹³C-glucoseresult and the HOMA. As can be seen, a significant correlation existsbetween the two indices. FIG. 7 shows the ROC plot for the 1.5-hour¹³C-glucose breath result. From this figure, a cutoff of 9.0 seems toprovide an optimal criterion for differentiating diabetic versusnon-diabetic.

Logistic regression based on clinical status as the dependent variable,and the 1.5-hour ¹³C breath test result, HOMA, age, BMI and sex asindependent variables, gave an R² value of 0.53. Only the 1.5-hour ¹³Cbreath test result gave a significant partial correlation coefficient.When the logistic regression was repeated using either the 1.5-hour ¹³Cbreath test result or the HOMA, but not both, in addition to the othervariables, only the ¹³C breath test result gave a significant partialcorrelation coefficient for the former regression. When the HOMA wasused, significant partial correlations were obtained for age and HOMA.The R² for the former regression was 48 and for the latter was 0.32.Thus the ¹³C breath test appears to be the strongest predictor ofdiabetic status.

Further variations and modification of the present invention will beapparent to those skilled in the art and are intended to be encompassedby the specification and claims appended hereto.

TABLE 1 Diagnostic Parameters for the Various Tests fpg WHO HOMA ¹³Cbreath ¹³C breath* sensitivity 43 62 67 73 75 specificity (97) (95) 6792 84 PPV (96) (92) 64 90 79 NPV 52 61 70 77 81 DA 64 74 67 79 80 fpg =fasting plasma glucose, ¹³C breath = test results from all 104 subjects,¹³C breath* = ¹³C breath test results from the subgroup where the HOMAwas calculated, PPV = positive predictive value, NPV = negativepredictive value, DA = diagnostic accuracy. Numbers in parenthesispresume normal subjects (1 for fpg and 2 for WHO) were incorrectlydiagnosed as being diabetic.

1. A method of diagnosing insulin resistance in a subject, said methodcomprising: a) collecting a first breath sample from said subject in afirst breath collection container; b) administering ¹³C-enriched glucoseto said subject; c) collecting a second breath sample from said subjectin a second breath container at a time point after administration ofsaid ¹³C-enriched glucose; d) measuring the ¹³CO₂ in each of said firstand second breath samples; and e) comparing the amount of ¹³CO₂in saidsecond breath sample with the amount of ¹³CO₂in said first breathsample, wherein the presence of less ¹³CO₂in said second breath samplecompared to normal control comparison values indicates insulinresistance in the subject wherein said comparison is made by choosing acutoff of receiver operated characteristics (ROC) values wherein thesensitivity and specificity are such as to maximize diagnostic accuracy.2. The method of claim 1, wherein said cutoff of ROC values is 8 to 10.3. The method of claim 2, wherein said ROC value is 9 to
 10. 4. A methodof diagnosing insulin resistance in a subject, said method comprising:a) collecting a first breath sample from said subject in a first breathcollection container; b) administering ¹³C-enriched glucose to saidsubject; c) collecting a second breath sample from said subject in asecond breath container at a time point after administration of said¹³C-enriched glucose; d) measuring the ¹³CO₂ in each of said first andsecond breath samples; e) comparing the amount of ¹³CO₂ in said secondbreath sample with the amount of 13CO2 in said first breath sample tocalculate a delta over baseline; and f) diagnosing insulin resistance ifthe calculated delta over baseline for the subject is less than astandard delta over baseline of a normal individual or of a typicaldiabetic individual.
 5. A method of diagnosing insulin resistance in asubject, said method comprising: a) collecting a first breath samplefrom the subject in a first breath collection container; b)administering ¹³C-enriched glucose to the subject; c) collecting asecond breath sample from the subject in a second breath container at afirst time point after administration of the ¹³C-enriched glucose; d)collecting a third breath sample from the subject in a third breathcontainer at a second time point after administration of the ¹³C-enriched glucose; e) measuring the ¹³CO₂ in each of the first, secondand third breath samples to generate a breath test curve; f) determininga blood glucose level of the subject prior to administration of the¹³C-enriched glucose and at the first and second time points afteradministration to generate a blood glucose test curve; g) diagnosinginsulin resistance if the area between the breath test curve and theblood glucose test curve is less than that of a standard individual orof a typical diabetic individual.